Heterodimerization with different Jun proteins controls c-Fos intranuclear dynamics and distribution.

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c-Fos intranuclear dynamics and distribution.

Cécile Malnou, Frederique Brockly, Cyril Favard, Gabriel Moquet-Torcy, Marc Piechaczyk, Isabelle Jariel-Encontre

To cite this version:

Cécile Malnou, Frederique Brockly, Cyril Favard, Gabriel Moquet-Torcy, Marc Piechaczyk, et al..

Heterodimerization with different Jun proteins controls c-Fos intranuclear dynamics and distribution..

Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2010, 285 (9), pp.6552-6562. �10.1074/jbc.M109.032680�. �hal-00455236�

Heterodimerization with Different Jun Proteins Controls c-Fos Intranuclear Dynamics and Distribution

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Received for publication, June 11, 2009, and in revised form, December 3, 2009Published, JBC Papers in Press, January 6, 2010, DOI 10.1074/jbc.M109.032680

Ce´cile E. Malnou^‡§¶1, Fre´de´rique Brockly^‡§¶, Cyril Favard^储, Gabriel Moquet-Torcy^‡§¶, Marc Piechaczyk^‡§¶2,3, and Isabelle Jariel-Encontre^‡§¶2,4

From the^‡Institut de Ge´ne´tique Mole´culaire de Montpellier, UMR5535, CNRS, 1919 route de Mende, 34293 Montpellier Cedex 5, the^§Universite´ Montpellier 2, Place Euge`ne Bataillon, 34095 Montpellier Cedex 5, the^¶Universite´ Montpellier 1,

34967 Montpellier Cedex 2, and the^储Institut Fresnel, CNRS UMR6133, Aix-Marseille Universite´, Ecole Centrale Marseille, Campus de Saint Je´roˆme, 13013 Marseille, France

The c-Fos proto-oncogenic transcription factor defines a multigene family controlling many processes both at the cell and the whole organism level. To bind to its target AP-1/12-O-tet- radecanoylphorbol-13-acetate-responsive element or cAMP- responsive element DNA sequences in gene promoters and exert its transcriptional part, c-Fos must heterodimerize with other bZip proteins, its best studied partners being the Jun pro- teins (c-Jun, JunB, and JunD). c-Fos expression is regulated at many transcriptional and post-transcriptional levels, yet little is known on how its localization is dynamically regulated in the cell. Here we have investigated its intranuclear mobility using fluorescence recovery after photobleaching, genetic, and bio- chemical approaches. Whereas monomeric c-Fos is highly mobile and distributed evenly with nucleolar exclusion in the nucleus, heterodimerization with c-Jun entails intranuclear redistribution and dramatic reduction in mobility of c-Fos caused by predominant association with the nuclear matrix independently of any binding to AP-1/12-O-tetradecanoylphor- bol-13-acetate-responsive element or cAMP-responsive ele- ment sequences. In contrast to c-Jun, dimerization with JunB does not detectably affect c-Fos mobility. However, dimeriza- tion with JunB affects intranuclear distribution with significant differences in the localization of c-Fos

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AP-1 complexes. This may affect the numerous physiopatholog- ical functions these transcription factors control.

The AP-1 transcriptional complex comprises a large family of dimeric transcription factors involved in the control of numerous physiological and pathological processes. These include among others cell proliferation, differentiation, apo- ptosis, responses to environmental cues, tumorigenesis, devel- opmental defects, and immune diseases (1–5). Its best studied components are the Fos (c-Fos, Fra-1, Fra-2, and FosB) and Jun (c-Jun, JunB, and JunD) family proteins, all of which necessitate dimerization via a leucine zipper (LZ)⁵to acquire transcrip- tional competence. Fos proteins can only heterodimerize with other AP-1 components, whereas Jun proteins can also homodimerize, even though heterodimerization with any of the Fos is favored (6, 7). Thanks to both the LZ and the adjacent basic DNA-binding domains (DBD), Fos

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c-Fos is the first discovered and best studied member of the Fos family (8, 9). It is constitutively expressed in a limited num- ber of tissues but is rapidly and transiently induced in many other cell types by a large variety of stimuli (9). In the latter case, c-Fos accumulation is controlled at the level of transcription, mRNA turnover, and protein stability (8, 10, 11). Moreover, c-Fos intracellular localization (see below) and transcriptional activity are tightly regulated (8). In particular, transcriptional activity can be enhanced via phosphorylation of various serines and threonines that may also participate in protein stabiliza- tion. The kinases include the MAPK p38, ERK1/2, and ERK5

*This work was supported by grants from the ARC, Association pour la Recherche sur le Cancer, the Agence Nationale de Recherches (ANR-08- BLAN-007– 01), the French Ligue Nationale Contre le Cancer, and by the CNRS.

□^S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

1Present address: UFR SVT, Universite´ Paul Sabatier, 31062 Toulouse and INSERM U563, Centre de Physiopathologie de Toulouse Purpan, 31024 Toulouse, France.

2Both authors contributed equally to this work.

3Supported in part by a Equipe Labelise´e grant from the French Ligue Natio- nale contre le Cancer. To whom correspondence may be addressed: IGMM, UMR5535, 1919 route de Mende, 34293 Montpellier Cedex 5, France. Tel.:

33-4-67-61-36-68; Fax: 33-4-67-04-02-31; E-mail: marc.piechaczyk@igmm.

cnrs.fr.

4To whom correspondence may be addressed: IGMM, UMR5535, 1919 route de Mende, 34293 Montpellier Cedex 5, France. Tel.: 33-4-67-61-36-68; Fax:

33-4-67-04-02-31; E-mail: [email protected], marc.piechaczyk@

igmm.cnrs.fr.

5The abbreviations used are: LZ, leucine zipper; FRAP, fluorescence recovery after photobleaching; CRE, cAMP-responsive element; TRE, 12-O-tetradecano- ylphorbol-13-acetate-responsive element; DBD, DNA-binding domain(s);

MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regu- lated kinase; STAT, signal transducers and activators of transcription; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline;

MNase, microccocal nuclease.

6552 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 9 •FEBRUARY 26, 2010

(12–25), where the role of ERK5 is disputed (26), as well as the ERK1/2-activated kinases Rsk1/2 (12, 14, 16, 22) and I␬B kinase (27). In contrast, c-Fos can be transcriptionally repressed by sumoylation at a specific lysine (28, 29). Interestingly, sumoy- lation of this lysine is antagonistic to a nearby transcription- activating phosphorylation (28).

Usually, c-Fos accumulates predominantly, if not exclu- sively, within the nucleus. However, it can also localize within the cytoplasm under certain conditions (17, 24, 25, 30, 31). Cytoplasmic c-Fos can even associate with the endoplasmic reticulum to activate phospholipid metabolism in a transcrip- tion activity-independent manner (32, 33). c-Fos intracellular localization is regulated by both intra- and extracellular signals with demonstrated roles for cAMP-dependent protein kinase A (34), the p38 MAPK (25), and the STAT3 transcription factor when ERK5 is inactivated (24). Consistent with this dual intra- cellular localization, we and others (24, 35, 36) have shown that c-Fos can shuttle between the nucleus and the cytoplasm. Entry into the nucleus is controlled by at least two nuclear localization signals: a conventional basic nuclear localization signal (35–37) that most likely utilizes the nuclear import receptor Imp␤1 (35, 36) and an unconventional nuclear localization signal located in the N-terminal moiety of the protein that requires the nuclear importin Transportin 1 (35, 36). Depending on the conditions, both Crm-1 exportin-dependent (24) and -independent (36) mechanisms involving different nuclear export signals are responsible for c-Fos nuclear exit. Interestingly, this involves primarily monomeric c-Fos and is inhibited upon heterodimer- ization with the Jun proteins (36). This indicated that dimeriza- tion is important, not only for the formation of active AP-1 transcription complexes but also to keep them in the nucleus where they play their transcriptional part. c-Fos nuclear reten- tion, however, varies according to its Jun partner. It is much stronger with c-Jun than with JunB or JunD. This correlates with the strength of interaction of the various c-Fos

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MATERIALS AND METHODS

Plasmids, Cloning, and Mutagenesis—Cloning and mutagen- esis were performed using standard PCR-based methods. All of the constructs were entirely sequenced. Plasmids and related information are available on request. pcDNA3-based expres- sion plasmids for wild type and mutant rat c-Fos and mouse JunB-FLAG and c-Jun-FLAG were previously described (16, 36). EGFP chimeras were obtained by insertion of either rat c-Fos or human Fra1 coding sequences into the pEGFP-C1 vec- tor (Clontech).

Cell Culture and Transfection—Conditions for culturing and transfecting human HeLa cells by the calcium phosphate co- precipitation technique were previously described (36). 3␮g of plasmid/10⁶cells was routinely used, and the transfection time was limited to 16 h to avoid protein overexpression.

Immunofluorescence and Confocal Microscopy—To set up the conditions of FRAP experiments on cells expressing levels of

EGFP-c-Fos or EGFP similar to that of endogenous c-Fos, we com- pared the expression level of transfected EGFP-c-Fos with that of endogenous c-Fos expressed upon 1 h of serum stimulation. To this aim, HeLa cells were either transfected for 16 h with the EGFP- or EGFP-c-Fos-encoding vector or stimulated with 20% serum for 1 h. The cells were fixed in 4% paraformaldehyde and permeabi- lized in the presence of 0.2% Triton X-100. They were then suc- cessively incubated with the H125 rabbit anti-c-Fos antibody (Santa Cruz Biotechnology), detecting equally c-Fos and EGFP- c-Fos and an Alexa 647-labeled anti-rabbit antibody (Molecular Probe). The nuclei were stained with Hoechst 33342, and cov- erslips were mounted in Permafluor. Cells expressing EGFP-c- Fos and emitting a fluorescence signal in the far red channel similar to that of cells expressing endogenous c-Fos were selected to set up the signal intensity range in the green channel.

This allowed us to select under the microscope cells expressing EGFP or EGFP-c-Fos at a level comparable with that of endog- enous c-Fos in the green channel. The far red fluorescence (Alexa 647) was monitored using a 560-nm long wavelength path filter, whereas EGFP fluorescence was monitored using a 505–550-nm wavelength band pass filter. Twelve-bit image acquisition was performed using a LSM510 meta microscope (Zeiss) equipped with a plan Apochromat 40

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FRAP Experiments—FRAP analyses were performed at 37 °C using a Zeiss LSM 510 Meta microscope equipped with a heat- ing chamber and a plan Apochromat 40

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Correction was carried out by dividing the value of the fluores- cence in the bleach area by that of another region in the nucleus far away from the bleached volume. Finally, fluorescence intensi- ties in the bleach area were normalized against the prebleach level of fluorescence intensity. Data fitting was performed using the Levenberg-Marquadt algorithm to determine both the mean half- time of fluorescence recovery (

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⫹A2

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whereI₀is the fluorescence intensity att

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recording) and with

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F_mob30⫽I_30s⫺I₀ 1⫺I0

(Eq. 2)

The choice of this model was conditioned to ac²statistical test showing the best fit for all of the data as compared with a mono- exponential or a two-dimensional diffusion under a Gaussian illumination model. This model was used without anya priori knowledge of either the geometry of the bleaching or the pro- cess of fluorescence recovery. It was therefore used as a model to compare mean half-times of fluorescence recovery. Finally,I₀ was determined by bleaching under the same experimental conditions as those described above for c-Fos-EGFP-express- ing cells after their fixation with 4% paraformaldehyde for 30 min at room temperature.I₀was set to 0.4.

Cell Fractionation in the Presence of Triton X-100—Cell frac- tionation procedures are those described in reference (39).

Approximately 10⁷cells were scrapped in PBS (150 mMNaCl, 10 mMsodium phosphate, pH 7) on ice, harvested by low speed centrifugation, resuspended in 200 ␮l of Buffer A (10 m^M Hepes, pH 7.9, 10 mMKCl, 1.5 mMMgCl₂, 0.34Msucrose, 10%

glycerol, 1 mMdithiothreitol, 1 Complete Mini protease inhib- itor mixture tablet (Roche Applied Science)/10 ml of buffer) and let on ice for 8 min. The cells were lysed in the presence of 0.15% Triton X-100 on ice for 4 min and subjected to centrifu- gation (3500 rpm for 5 min). The supernatant (S) contained both the cytoplasmic and the soluble nuclear fractions. The nuclei were then washed once in 0.15% Triton X-100-contain- ing buffer A and recentrifuged at 3500 rpm for 5 min. After resuspension in 200␮l of Buffer B (3 mMEDTA, 0.2 mMEDTA, 1 mMdithiothreitol, 1 Complete Mini protease inhibitor mix- ture tablet/10 ml of buffer), the nuclei suspension was left on ice for 30 min for membrane disruption and then centrifuged at 4000 rpm for 5 min. The supernatant corresponded to the wash fraction (W). The pellet (P) was subjected to a round of washing in buffer B and centrifugation and finally resuspended in Lae- mmli electrophoresis loading buffer. The various fractions were then submitted to immunoblotting analyses as previously described (36).

Immunoprecipitation and Immunoblotting Experiments—

Immunoprecipitations were performed as described in Ref. 28.

10⁷cells were lysed in 600 ␮l of radioimmune precipitation assay buffer (50 mMTris-HCl, pH 8.0, 150 mM NaCl, 0.02%

NaN₃, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 Complete Mini protease inhibitor mixture tablet/10 ml of buffer). To immunoprecipitate FLAG-tagged proteins, 200␮l of lysates were incubated for 3 h in the presence of 30␮l of anti-FLAG M2 affinity gel from Sigma. After centrifugation of cell extract-containing suspensions, the supernatants were col- lected, whereas the pellets were subjected to five cycles of wash- ing in radioimmune precipitation assay buffer and centrifuga- tion. For immunoblotting analyses, total extracts, supernatants, and immunoprecipitated fractions were electrophoresed through 12% polyacrylamide gels containing SDS and electro- transferred onto polyvinylidene difluoride membranes. Immu- nodetections were carried out with appropriate dilutions of the various following primary antibodies: rabbit anti-c-Fos H125

(sc-7202), rabbit anti-c-Jun H79 (sc-1694), and goat anti-JunB (sc-46G) from Santa Cruz Biotechnology. We also used rabbit antisera directed to topoisomerase I (kind gift from Dr J. Soret).

The anti-Phax mouse monoclonal and the anti-lamin rabbit polyclonal antibodies were kindly provided by Drs. D. Lener, and H. Wodrich, respectively. Secondary horseradish peroxi- dase-coupled antibodies were either from Santa Cruz Biotech- nology (sc-2313 anti-rabbit and sc-2033 anti-goat horseradish peroxidase conjugates) or from Sigma (A-9044 anti-mouse horseradish peroxidase conjugate). Chemoluminescence was detected with the Chemoluminescence reagent Plus kit from PerkinElmer Life Sciences using Biomax XAR Kodak films.

Chromatin and Nuclear Matrix Fractionation: Biochemical and Microscope Analyses—For analysis of endogenous c-Fos, HeLa cells were grown for 4 days without any change of culture medium and stimulated for 1 h by the addition of 20% fresh serum. For analysis of exogenous c-Fos, HeLa cells were trans- fected with a vector for EGFP-c-Fos alone or in combination with vectors for either c-Jun or JunB in a 1:2 ratio. Our fraction- ation procedure combined the methods described in Refs. 40 and 41. For immunoblotting analyses, 4

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NaCl, 3 mMMgCl_2,1 mMEGTA). After centrifugation, the pel- lets were rinsed once with 1 ml of CSK buffer and recentrifuged, and cell lysis was allowed to proceed for 10 min at 0 °C (10⁷ cells/ml) in CSK buffer containing 0.5% Triton X-100, 1␮g/ml leupeptin, 1␮g/ml aprotinin, 1␮g/ml pepstatin A/ml, 25␮g/ml 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and 400 units/ml RNasin. Half of each sample was mixed with Lae- mmli sample buffer to constitute the total cell extract (T). The nuclei of the other half were pelleted by centrifugation at 5000 rpm for 2 min at 4 °C. The supernatants (S1), which contained solubilized cytoplasmic and nuclear proteins, were collected.

The nuclei were washed once with 200␮l of the above ice-cold lysis buffer and pelleted by centrifugation. Centrifugation supernatants corresponded to the wash fractions (W). The nuclei were then treated with microccocal nuclease (MNase;

Roche Applied Science) to remove chromatin- and DNA- bound proteins. To this aim, they were resuspended (10⁷ nuclei/ml) in ice-cold CSK containing 0.5% Triton X-100, pro- tein, and RNase inhibitors as above, 10 mMCaCl₂ and 200 units/ml of MNase. After a 10-min incubation at 30 °C, they were centrifuged, and supernatants (S2) were collected. After resuspension in CSK buffer containing 2MNaCl and protease and RNase inhibitors as above, they were let for 5 min at 0 °C to remove the remaining DNA and histones. Pellets (P) containing nuclear matrix and associated proteins were collected by cen- trifugation. Supernatants (S3) contained nuclear proteins solu- ble in 2MNaCl. Fluorescence microscope analyses of cells were conducted in parallel. To this aim, the coverslips were placed in culture dishes before cell seeding and processed separately at the time of biochemical cell/nucleus fractionation following the same steps as those described above. After rinsing in the pres- ence of PBS, the cells were subsequently fixed in the presence of 4% paraformaldehyde (i) before lysis, (ii) after Triton X-100 cell lysis, (iii) after MNase treatment, and (iv) after 2MNaCl treat- ment. When necessary (non-Triton X-100 lysed cells), an addi-

tional permeabilization treatment with 0.2% Triton X-100 in PBS for 5 min at room temperature was added before micro- scopic analysis. EGFP-c-Fos was followed up using direct fluo- rescence, whereas endogenous c-Fos required indirect fluores- cence analysis using the sc-52 (Santa Cruz Biotechnology), anti-c-Fos antibody, and an Alexa 488-labeled anti-rabbit antibody (Molecular Probes). The nuclei were stained with

Hoechst 33342 at a concentration of 0.2␮g/ml. The coverslips were mounted in Permafluor.

Fluorescence Analysis of Fixed Cells—Observations were per- formed using a Leica DMRA micro- scope equipped with a 63

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All of the images were acquired using the same exposure time to compare cells with equivalent c-Fos levels.

RESULTS

Monomeric c-Fos Is Highly Mobile in the Nucleus—To study c-Fos intranuclear mobility, we used the technique of FRAP (42– 44) on live human HeLa cells expressing an EGFP-c-Fos fusion protein. This chimera, in which c-Fos is fused to the C terminus of EGFP, has been shown to retain the follow- ing properties of wild type c-Fos:

dimerization with all Jun family members, short half-life and pro- teasome-dependent degradation, quantitative nuclear accumulation, and nucleo-cytoplasmic shuttling inhibited by c-Jun (16, 36). We also verified that it transactivates an AP-1-luciferase reporter gene to a similar extend as wild type c-Fos (data not shown).

When expressed alone in tran- sient transfection assays, c-Fos and EGFP-c-Fos are essentially mo- nomeric (36) and predominantly nuclear despite their active nu- cleo-cytoplasmic shuttling (36).

Moreover, they show a typical dif- fuse distribution with nucleolar exclusion, as assayed both by indi- rect immunofluorescence (36) or by direct visualization of EGFP-c-Fos in living cells (Fig. 1,AandB). We used FRAP (n

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endogenous c-Fos expressions. HeLa cells were either stimulated for 1 h with 20% serum (left panel) or trans- fected for 16 h with the EGFP-c-Fos-encoding plasmid (right panel). After cell fixation and permeabilization, c-Fos and EGFP-c-Fos were stained with a rabbit anti-c-Fos antiserum followed by an Alexa 647-labeled anti- rabbit antiserum. EGFP-c-Fos-expressing cells emitting a far red signal close to that of cells expressing endog- enous c-Fos were selected and observed in the green channel to set up the green fluorescence intensity range usable in FRAP experiments.B, FRAP experiment. Asynchronously growing HeLa cells were transfected with expression plasmids encoding EGFP-c-Fos or EGFP, and FRAP experiments were carried out as described under

“Materials and Methods.” A typical experiment with EGFP-c-Fos is presented.Arrowsindicate the bleached area, before, during, and after the bleach.C, FRAP data. Typical fluorescence recovery curves are presented for both EGFP- and EFGP-c-Fos-expressing cells.⬍t¹⁄2⬎andFmob30calculated from 20 FRAP experiments are given. They include standard deviations.D, immunoblotting analysis of cell fractions. Cells transfected as inA were lysed in the presence of Triton X-100, and soluble (S), wash (W), and nonsoluble (NS) fractions were prepared and analyzed by immunoblotting as described under “Materials and Methods.” Phax and topoi- somerase I (Topo 1) were used as internal controls of soluble and nonsoluble proteins, respectively.

induced upon growth factor stimulation (8, 9) and compared exogenous EGFP-c-Fos and EGFP abundance in transfected HeLa cells with that of endogenous c-Fos in HeLa cells stimu- lated for 1 h by serum using standardized fluorescence acquisi- tion procedures, as detailed under “Materials and Methods”

and shown Fig. 1A. Typical FRAP experiments are presented in Fig. 1B. Mean half-time fluorescence recovery (

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1C). The

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To complement these FRAP studies, we performed fraction- ation experiments on transfected HeLa cells, comparing the distri- bution of EGFP-c-Fos with that of c-Fos in Triton X-100-soluble and -insoluble fractions. Monomeric EGFP-c-Fos and monomeric c-Fos were principally found in the soluble fraction (S) together with the nucleoplasmic protein Phax (46) taken as a control (Fig.

1D; also see Fig. 2,DandE). Sometimes, a minor proportion of monomeric EGFP-c-Fos or c-Fos remained in the insoluble frac- tion (NS), which was monitored using chromatin-bound topoi- somerase I (47) (Fig. 1D; also see Fig. 2,DandE). Thus, the high mobility of monomeric c-Fos in the nucleus correlates with its lack of strong interaction with intranuclear structures.

c-Jun Reduces c-Fos Intranuclear Mobility—We next tested whether dimerization with c-Jun altered c-Fos intranuclear mobility. Fos and Jun family members undergo dimerization/

dedimerization cyclesin vivo. Nevertheless, they interact for at least several minutes (48). Thus, the mobility of Fos

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Strikingly, co-expression of c-Jun led to a dramatic intranu- clear redistribution of EGFP-c-Fos, which became less homog- enous showing irregular areas of accumulation (Fig. 2A). We then performed FRAP experiments on HeLa cells, where EGFP- c-Fos was expressed together with varying amounts of c-Jun.

Interestingly, the mobile fraction of EGFP-c-Fos progressively

diminished as the amount of c-Jun increased. At 2- and 5-fold excesses of c-Jun expression plasmid, theF_mob30of EGFP-c-Fos was

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We noted that only 60% of c-Fos remained immobile during the time course of the experiments, even though co-immuno- FIGURE 2.Reduced intranuclear mobility of c-Fos in the presence of c-Jun.

A, alteration of intranuclear distribution of EGFP-c-Fos in the presence of c-Jun. HeLa cells were transfected with EGFP-c-Fos in the absence or in the presence of a 2-fold excess of c-Jun expression plasmid and analyzed by con- focal microscopy on living cells.B,F_mob30of EGFP-c-Fos in the presence of varying amounts of c-Jun. HeLa cells were co-transfected with c-Jun and EGFP-c-Fos expression plasmids in the indicated ratios. FRAP experiments were performed as in Fig. 1A.F_mob30values were calculated 30 s after the end of the bleach from 20 experiments for each condition and presented as his- tograms. Theerror barsindicate standard deviations.C, FRAP experiment.

Asynchronously growing HeLa cells were transfected with expression plas- mids encoding EGFP with or without a 2-fold excess of c-Jun vector. Each curve corresponds to the averages of 20 FRAP experiments.DandE, cell fractionation experiments. Fractionation experiments were carried out as in Fig. 1Cusing HeLa cells co-transfected with expression plasmids for (i) EGFP- c-Fos in the absence or in the presence of a 2-fold excess of c-Jun expression plasmid (D) or (ii) c-Fos in the absence or in the presence of a 2-fold excess of c-Jun expression plasmid (E).S, soluble;W, wash;NS, nonsoluble.

precipitation experiments indicated quantitative association of c-Fos with c-Jun. Thus, 40% of dimers appeared mobile for the 30-s post-bleach. This likely reflected saturation of the nuclear binding sites for c-Fos

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Reduced c-Fos Intranuclear Mobility upon Heterodimeriza- tion with c-Jun Does Not Require DNA Binding—One obvious explanation for the reduction of c-Fos intranuclear mobility by c-Jun would be binding of heterodimers to genomic AP-1 motifs. To test this possibility, we compared the behavior of different heterodimers composed of (i) wild type EGFP-c-Fos or EGFP-c-Fos mutated in either the leucine zipper or the DNA-binding domain and (ii) wild type or leucine zipper mutants of c-Jun (Fig. 3A). The dimerization mutants of EGFP- c-Fos were either deleted of the LZ (EGFP-c-Fos

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For c-Jun, we used only a dimerization mutant lacking the LZ

(c-Jun

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Despite the presence of a 2-fold excess of the c-Jun expres- sion plasmid, EGFP-c-FosVAV and EGFP-c-Fos

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6C. E. Malnou, F. Piechaczyk, and I. Jariel-Encontre, unpublished data.

FIGURE 3.Role of heterodimerization with c-Jun and binding to genomic AP-1 DNA sequences on c-Fos intranuclear dynamics.A, structures of wild type and mutant c-Fos and c-Jun proteins.B,F_mob30of wild type and mutant EGFP-c-Fos proteins in the presence of wild type and mutant c-Jun. Co-transfections were conducted in the presence of 2-fold more c-Jun expression plasmids than vectors for either wild type or mutant EGFP-c-Fos. FRAP experiments were conducted as in Fig. 1B.F_mob30are the averages of 6 –20 experiments. The given values include standard deviations.C, localization of wild type and mutant EGFP-c-Fos in the presence of wild type c-Jun. Transfection conditions were as inB. Confocal microscopy was conducted on living cells.D, dimerization of EGFP-c-Fos mutants with c-Jun.

Transfection conditions were as inB. Co-immunoprecipitations were performed with the anti-FLAG antibody, because c-Jun constructs contained a FLAG tag at the C terminus, and immunoblotting experiments were conducted with the indicated antibodies as described under “Materials and Methods.”E, cell fractionation experi- ments. Transfection conditions were as inB, and cell fractionations were conducted and analyzed as in Fig. 1C.T, total cell extract;SN, supernatant;S, soluble;W, wash;

NS, nonsoluble;IP, immunoprecipitation.

sequences is the primary event responsible for this c-Jun- mediated reduction of c-Fos mobility. Furthermore, cell fractionation experiments showed that EGFP-c-Fos

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JunB Does Not Affect c-Fos Intranuclear Mobility—It seemed important to test whether another dimerization partner could have the same effect on c-Fos intranuclear mobility. JunB is also a highly documented c-Fos partner (6, 15, 48, 53). Therefore, we analyzed its effects on EGFP-c-Fos intracellular localization and mobility as we did with c-Jun. Under conditions of quanti- tative association with JunB, as assayed by co-immunoprecipi- tation (Fig. 4A), EGFP-c-Fos was redistributed within the nucleus, as seen upon dimerization with c-Jun, albeit with a

reproducible significantly different distribution (Fig. 4B; com- pare with Fig. 2A). FRAP experiments showed both an initial recovery of fluorescence (Fig. 4C) and anF_mob30(Fig. 4D) sim- ilar for JunB

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Fra-1 Intranuclear Distribution and Mobility Are Affected by c-Jun and JunB in a Manner Similar to c-Fos—We then tested whether c-Jun and JunB similarly influenced the behavior of the Fos family protein Fra-1, because endogenous c-Jun and JunB FIGURE 5.Alteration of Fra-1 intranuclear distribution and mobility by c-Jun and JunB.A, dimerization of EGFP-Fra-1 with c-Jun and JunB. Het- erodimerization assays were conducted as in Fig. 3Dusing HeLa cells trans- fected with plasmids for either c-Jun-FLAG⫹EGFP-Fra-1 or JunB-FLAG⫹ EGFP-Fra-1 in a ratio of 2.B, intracellular localization of EGFP-Fra-1 in the presence of c-Jun and JunB in a 2-fold excess. Intracellular localization was assessed on living cells by confocal microscopy analysis of HeLa cells trans- fected as inA.C, FRAP experiments. FRAP experiments were conducted in HeLa cells transfected with expression plasmids for either EGFP-Fra1 or EGFP- Fra1 and a 2-fold excess of JunB- or c-Jun plasmid. Thecurvescorrespond to the averages of 10 –20 FRAP experiments.D,F_mob30of EGFP-Fra-1 in the pres- ence of c-Jun and JunB. Transfection conditions were as inA.F_mob30were calculated from more than 10 FRAP experiments.E, fractionation experi- ments. Fractionation and analyses of HeLa cells transfected as inAwere con- ducted as in Fig. 1C.T, total cell extract;SN, supernatant;IP, immunoprecipi- tation;S, soluble;W, wash;NS, nonsoluble.

FIGURE 4.Effect of JunB on c-Fos intranuclear distribution and mobility.

A, heterodimerization of JunB-FLAG with EGFP-c-Fos. The expression plasmid for EGFP-c-Fos was transfected in HeLa cells in the presence of a 2-fold excess of JunB-FLAG construct. Co-immunoprecipitations were conducted with the anti-FLAG antibody as in Fig. 3D. The antibodies used in immunoblotting analyses are indicated.B, intranuclear distribution of EGFP-c-Fos in the pres- ence of JunB. EGFP-c-Fos localization in the presence of JunB-FLAG was assessed by confocal microscopy on living cells. The JunBversusEGFP-c-Fos expression plasmid ratio was of 2.C, FRAP experiments. FRAP experiments were conducted in HeLa cells transfected with expression plasmids for either EGFP-c-Fos or EGFP-c-Fos⫹JunB. In the latter case, the plasmid ratio was 2.

Thecurvescorrespond to the averages of 20 FRAP experiments in each case.

D, mobility of EGFP-c-Fos in the presence of different amounts of JunB. HeLa cells were transfected in the presence of different ratios of JunBversusEGFP- c-Fos plasmids as indicated.F_mob30were calculated from 15–20 FRAP exper- iments in each case.E, cell fractionation experiments. Cell fractionation exper- iments of cells transfected with plasmids encoding JunB and EGFP-c-Fos in a ratio of 2 were conducted and analyzed as in Fig. 1D.T, total cell extract;SN, supernatant;IP, immunoprecipitation;S, soluble;W, wash;NS, nonsoluble.

are known to interact with endogenous Fra-1 in a variety of situations (6, 48, 53). EGFP-Fra-1 quantitatively dimerizes with ectopic c-Jun and JunB in HeLa cells (Fig. 5A). Interestingly, monomeric EGFP-Fra-1 was weakly cytoplasmic, which was no longer seen in the presence of co-transfected c-Jun and JunB (Fig. 5B). Like for c-Fos, FRAP analyses indicated that Fra-1 intranuclear mobility was dramatically reduced when co-ex- pressed with c-Jun but was unaffected by co-expression of JunB (Fig. 5C). The mobile fraction of Fra-1 decreased to

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Differential Association of c-Fos

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First, we conducted classical biochemical fractionation ex- periments (see “Materials and Methods”) followed by immuno- blotting assays. In these experiments: (i) S1 corresponded to both cytoplasmic and nuclear proteins solubilized upon cell lysis in the presence of 0.5% Triton X-100, (ii) S2 corresponded to the chromatin proteins released upon subsequent extensive DNA hydrolysis by MNase, and (iii) S3 corresponded to the remnant of chromatin proteins and DNA solubilized after addi- tional high salt (2MNaCl) treatment, and (P) corresponded to the nuclear-matrix-containing fraction. Fractionation patterns of (i) the nucleosoluble Phax protein, (ii) the weakly chromatin- associated HCF-1 protein (54), (iii) tightly chromatin-associ- ated histone H3, and (iv) the nuclear lamina-constituting lamin A/C proteins confirmed the efficiency of the procedure (Fig. 6).

In contrast to EGFP-c-Fos expressed alone, which localized predominantly in the S1 fraction, EGFP-c-Fos

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However, a very minor fraction of EGFP-c-Fos was found in the P fraction. This may be due to dimerization of the protein with

endogenously expressed AP-1 pro- tein, although partial association of the monomer with the nuclear matrix cannot be formally excluded.

Differing from EGFP-c-Fos

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S1, S2, and P, with approximately half of them in the P fraction. We also analyzed the distribution of endogenous c-Fos in HeLa cells stimulated with 20% serum for 1 h.

Interestingly, it behaves as EGFP-c- Fos

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of c-Fos associates with JunB in com- parable serum stimulation experi- ments (48).

We then complemented these biochemical experiments by microscopic analyses (Fig. 7) of cells subjected to the same suc- cession of treatments (Triton X-100 solubilization, DNA hy- drolysis by MNase, and high salt washing) as above. Hoescht 33342 staining revealed large and total elimination of DNA after MNase and high salt treatment, respectively. EGFP-c- Fos

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This was consistent with intranuclear retention caused by dimerization with endogenous AP-1 proteins. Taken together, these observations were coherent with the fact that abolishing the DNA binding ability of c-Fos altered neither intranuclear mobility (Fig. 3B) nor its subcellular distribution (Fig. 3D) when dimerized with c-Jun. The fact that EGFP-c-Fos

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DISCUSSION

This study reveals a novel level of regulation operating on c-Fos, namely the differential control of its intranuclear mobil- ity and distribution by its Jun partners. This regulation also applies to at least another member of the Fos family, Fra-1.

Our data indicate that EGFP-c-Fos does not diffuse as freely as EGFP in FRAP experiments, indicating weak interactions between monomeric c-Fos and some nuclear structures. Inter- estingly, heterodimerization with c-Jun results in a dramatic reduction of c-Fos intranuclear mobility. Such a mobility FIGURE 6.Cell and subnuclear fractionation.HeLa cells were (i) transfected with the EGFP-c-Fos vector in the

absence or in the presence of an equivalent vector for either c-Jun or JunB in a 1:2 ratio or (ii) simply stimulated with 20% serum for 1 h. They were then fractionated as described under “Materials and Methods.” Equivalent amounts of each fraction were loaded in each lane for immunoblotting analysis. The names of the various proteins assayed are indicated in the figure.T, total cell extract;S1, Triton X-100-soluble fraction;W, wash;S2, soluble fraction after MNase digestion;S3, soluble fraction after 2MNaCl treatment;P, nuclear matrix fraction.

change is not the result of dimerizationper sebut is selective because JunB does not detectably alter c-Fos intranuclear dynamics (as visualized by FRAP) under conditions where c-Fos is quantitatively associated with JunB (as shown by co- immunoprecipitation). Although the FRAP data presented in Figs. 2– 4 were obtained in human epithelial HeLa cells, similar differential effects of c-Jun and JunB were observed in Balb/C 3T3 mouse embryo fibroblasts (supplemental Fig. S1), indicat- ing that immobilization of c-Fos by c-Jun is neither cell- nor species-specific.

It is most often considered that reduced intranuclear mobil- ity of transcription factors is largely determined by binding to target DNA sequences because disabled DNA-binding domain mutants of proteins such as NF-␬B p65 (55), Acep1 (56), inter- feron regulatory factor 8 (57), the androgen receptor (58, 59), or the glucocorticoid receptor (60) are more mobile than their wild type counterparts. However, immobilization of c-Fos by c-Jun is not primarily mediated by binding to AP-1/TRE or CRE target sequences (also see below) because EGFP-c-Fos and EGFP-c-Fos⌬DBD behave similarly in FRAP experiments, when quantitatively heterodimerized with c-Jun. Strengthen- ing the idea of a DNA-independent mechanism responsible for c-Fos mobility reduction, cell fractionation experiments (Figs. 6 and 7) showed that c-Fos

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There may be an apparent paradox between the well known strong transcriptional activity of c-Fos

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Very interestingly, Andre´s and co-workers (63, 64) have recently reported that c-Fos can associate with the nuclear matrix component lamin A/C. This was proposed to constitute an AP-1 suppression mechanism. Although the authors did not address c-Fos mobility directly, their data are consistent with our cell fractionation experiments showing interaction with the nuclear matrix. From in vitro pull-down experiments, these authors hypothesized that c-Fos would interact with lamin A/C via its leucine zipper at the detriment of dimerization with its Jun partners (63). Although not questioning the principle of AP-1 inactivation by lamin A/C, our data, however, argue against LZ- mediated sequestration of monomeric c-Fos, because we clearly show that (i) monomeric c-Fos is highly mobile and quantitatively found in the soluble protein fraction and (ii) only dimeric c-Fos is found associated with the nuclear matrix.

The behavioral differences between c-Fos

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localization in fractionated cells.These experiments were carried out in parallel with those presented in Fig. 6 with HeLa cells seeded and grown on coverslips. Endogenous c-Fos was detected using sequentially the sc52 rabbit anti-c-Fos antibody and an Alexa 488-labeled anti-rabbit secondary antibody.

The nuclei were stained with Hoechst 33342. All of the pictures in the EGFP channel were acquired using the same time exposure.